We are developing a novel surgical technique that utilizes a two-photon laser to create transections, as narrow as 1 um in diameter, at any specified depth in the brain without damaging superficial or adjacent neuronal tissue. As we demonstrate in our preliminary data, we can already successfully perform this technique. These two-photon transections will be used to sever the neuropil, including somata and axonal and dendritic processes, to explore new possibilities for interrupting the initiation and propagation of neocortical epilepsy. Two-photon laser excitation is based on the principle that the energy of two photons arriving simultaneously at a point can excite a molecule in an extremely localized point in space. Although two-photon lasers have revolutionized Physics, Chemistry and Biology, they has never been applied as a surgical tool. The project will be a collaboration between Theodore Schwartz MD, an epilepsy surgeon and expert in in vivo optical imaging of neocortical epilepsy, and Rafael Yuste MD, Ph.D., a world expert in two-photon imaging and neocortical slice physiology. As co-investigators with a history of successful collaboration, the PIs will first optimize the two-photon lesioning technique in several models of in vitro rodent neocortical epilepsy, including superfusion with bicuculline and 4-aminopyridine. Epileptiform propagation will be monitored with voltage-sensitive dyes and tissue destruction assessed with calcium imaging and biocytin labeling of single cells followed by Neurolucida reconstructions and standard anatomical techniques. Two-photon transections will then be performed in both acute and chronic in vivo models of rodent neocortical epilepsy. Epilepsy propagation will be monitored using optical recording of intrinsic signals and tissue destruction assessed with the use of a "gene gun" for fluorescent labeling. The ability to create two-photon transections with a spatial resolution of 1 um will then allow us to address novel questions previously limited by technical restrictions, such as whether epileptiform initiation or propagation occurs preferentially in specific cortical layers in vivo. In addition, we will examine the optimal spacing and orientation of transections to eliminate abnormal excitability while maximizing the integrity of the cortical circuitry. The incredible spatial resolution of the two-photon transections will minimize surrounding tissue damage thereby permitting the possibility of a surgical cure for patients with neocortical epilepsy arising from function areas of brain who may not previously have been considered for surgery.